Immunoglobulin (Ig) genes are unique in that they are subject to three different types of gene alterations to achieve a fully functional humoral immune response. During early B cell development in the bone marrow (BM), V(D)J or VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase. Following exit from the bone marrow, B cells migrate to the secondary lymphoid organs where they undergo somatic hypermutation (SHM) and class switch recombination (CSR) that is initiated in both cases by activation induced deaminase (AID). Current paradigms describe a strict partition VDJ recombination to the BM and CSR and SHM to the secondary lymphoid organs. However, several lines of evidence have emerged over time suggesting that CSR and SHM can occur at low frequency at very early stages of B cell ontogeny. We have developed new data showing that robust CSR can be induced in Rag1-/- or Mb1-/- pro-B cells in ex vivo cultures expanded with IL7 and in vivo following injection of mice with LPS. We observe that in pro-B cells prior to VDJ or VJ joining, CSR inducers can stimulate the expression of AID and germline transcripts that are critically required for CSR. We find that CSR can be induced in a series of Abelson transformed cell lines followed by the induction of VDJ joining, thereby providing important tools to better analyze these observations. Strikingly, we find that AID and Rag gene expression can overlap which may account for the bulk of pro-B and pre-B cell chromosomal translocations in human leukemias and lymphomas. Based on these intriguing new studies we propose to more fully characterize CSR during early B cell development. We have carried out in vitro studies and show directly that VDJ joining can occur in CSR experienced cells using Abelson cell lines. We propose to determine whether secondary Ig isotypes detected in the MT mouse may derive from CSR in bone marrow pro-B cells. The in vivo frequency of AID+ and IgE+ pro-B cells induced in Rag1-/- or Mb1-/- pro- B cells will be determined using two mouse reporter strains. Enrichment of AID+-EYFP pro-B cells will enable us to characterize CSR and to determine whether VDJ repertoires are selected in this process. Finally we will determine whether Igh-myc junction fragments associated with high frequency T12;15 translocations in mature B cells are detectable in AID+ pro-B cells. These studies will form the basis for new insights regarding development of humoral immunity and early B cell oncogenesis. Public Health Relevance: B lymphocyte development in the bone marrow is characterized by DNA rearrangements termed VDJ joining, leading to expression of IgM antibodies. Based on new evidence that a switch from IgM to other immunoglobulin isotypes can occur prior to VDJ joining we will study the temporal ordering of antibody gene rearrangements and their relationship to immunodeficiency and autoimmune diseases.